Prevention of radiation-induced bystander effects by agents that inactivate cell-free chromatin released from irradiated dying cells.

Radiation-induced bystander effect (RIBE) is a poorly understood phenomenon wherein non-targeted cells exhibit effects of radiation. We have reported that cell-free chromatin (cfCh) particles that are released from dying cells can integrate into genomes of surrounding healthy cells to induce DNA damage and inflammation. This raised the possibility that RIBE might be induced by cfCh released from irradiated dying cells. When conditioned media from BrdU-labeled irradiated cells were passed through filters of pore size 0.22 µm and incubated with unexposed cells, BrdU-labeled cfCh particles could be seen to readily enter their nuclei to activate H2AX, active Caspase-3, NFκB, and IL-6. A direct relationship was observed with respect to activation of RIBE biomarkers and radiation dose in the range of 0.1-0 Gy. We confirmed by FISH and cytogenetic analysis that cfCh had stably integrated into chromosomes of bystander cells and had led to extensive chromosomal instability. The above RIBE effects could be abrogated when conditioned media were pre-treated with agents that inactivate cfCh, namely, anti-histone antibody complexed nanoparticles (CNPs), DNase I and a novel DNA degrading agent Resveratrol-copper (R-Cu). Lower hemi-body irradiation with γ-rays (0.1-50 Gy) led to activation of H2AX, active Caspase-3, NFκB, and IL-6 in brain cells in a dose-dependent manner. Activation of these RIBE biomarkers could be abrogated by concurrent treatment with CNPs, DNase I and R-Cu indicating that activation of RIBE was not due to radiation scatter to the brain. RIBE activation was seen even when mini-beam radiation was delivered to the umbilical region of mice wherein radiation scatter to brain was negligible and could be abrogated by cfCh inactivating agents. These results indicate that cfCh released from radiation-induced dying cells are activators of RIBE and that it can be prevented by treatment with appropriate cfCh inactivating agents.

Hydrogen as a new class of radioprotective agent.

It is well known that most of the ionizing radiation-induced damage is caused by hydroxyl radicals (·OH) follows radiolysis of H2O. Molecular hydrogen (H2) has antioxidant activities by selectively reducing ·OH and peroxynitrite(ONOO-). We firstly hypothesized and demonstrated the radioprotective effect of H2 in vitro and in vivo, which was also repeated on different experimental animal models by different departments. A randomized, placebo-controlled study showed that consumption of hydrogen-rich water reduces the biological reaction to radiation-induced oxidative stress without compromising anti-tumor effects. These encouraging results suggested that H2 represents a potentially novel preventative strategy for radiation-induced oxidative injuries. H2 is explosive. Therefore, administration of hydrogen-rich solution (physiological saline/pure water/other solutions saturated with H2) may be more practical in daily life and more suitable for daily consumption. This review focuses on major scientific and clinical advances of hydrogen-rich solution/H2 as a new class of radioprotective agent.

Implications of using the fluorescent probes, dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate, for the detection of UVA-induced reactive oxygen species.

During investigation of UVA-induced oxidative stress in HaCaT keratinocytes with dihydrorhodamine 123 (DHR123) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), exaggerated baseline values were observed within control samples, suggesting a mechanism of probe oxidation and subsequent change in fluorescence intensity (FI) independent of cellular ROS generation. The effects of diluent, UVA pre-treatment and loading protocols upon the FI of the probes have therefore been investigated. The study confirmed the capacity of Dulbecco's Modified Eagle's Medium (DMEM) to confer fluorescence intensity changes in both probes, most notably DCF-DA. In addition, UVA pre-treatment compromises the effectiveness of DHR123 and DCF-DA to detect ROS generated in a cell-free system. In vitro data shows a greater UVA-induced FI increase in HaCaT cells loaded with probe before rather than after UVA treatment. This study has important implications for future research, the understanding of previous studies and associated confounding effects using DHR123 and DCF-DA as ROS sensitive probes.

Charged liposome affects the translation and folding steps of in vitro expression of green fluorescent protein.

The role of the charged liposome on the in vitro expression of green fluorescent protein (GFP) was investigated, focusing on its elemental steps such as transcription, translation and folding. The total GFP expression was enhanced to 145% when a neutral liposome (POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline) was added externally to a cell-free translation system. On the contrary, the addition of the charged liposome composed of POPC with anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) or cationic stearyl amine (SA) inhibited the total GFP expression, depending on the surface charge density of liposome. In transcription, the RNA synthesis was enhanced regardless of the variation of the surface charge, indicating that transcription was enhanced due to the stabilization of RNA structure by its hydrophobic interaction with liposome. Translation was inhibited by cationic liposome although it was enhanced by anionic liposome and neutral liposome. On the other hand, the folding was not inhibited in the presence of neutral liposome, whereas anionic liposome and cationic liposome inhibited the folding in proportion to the their surface charges, suggesting that the total GFP expression was controlled by a charged liposome in the translation step and folding step.

Alpha-anomeric deoxynucleotides, anoxic products of ionizing radiation, are substrates for the endonuclease IV-type AP endonucleases.

Alpha-anomeric 2'-deoxynucleosides (alphadN) are one of the products formed by ionizing radiation (IR) in DNA under anoxic conditions. Alpha-2'-deoxyadenosine (alphadA) and alpha-thymidine (alphaT) are not recognized by DNA glycosylases, and are likely removed by the alternative nucleotide incision repair (NIR) pathway. Indeed, it has been shown that alphadA is a substrate for the Escherichia coli Nfo and human Ape1 proteins. However, the repair pathway for removal of alphadA and other alphadN in yeast is unknown. Here we report that alphadA when present in DNA is recognized by the Saccharomyces cerevisiae Apn1 protein, a homologue of Nfo. Furthermore, alphaT is a substrate for Nfo and Apn1. Kinetic constants indicate that alphadA and alphaT are equally good substrates, as a tetrahydrofuranyl (THF) residue, for Nfo and Apn1. Using E. coli and S. cerevisiae cell-free extracts, we have further substantiated the role of the nfo and apn1 gene products in the repair of alphadN. Surprisingly, we found that bacteria and yeast NIR-deficient mutants are not sensitive to IR, suggesting that DNA strand breaks with terminal 3'-blocking groups rather than alphadN might contribute to cell survival. We propose that the novel substrate specificities of Nfo and Apn1 play an important role in counteracting oxidative DNA base damage.

Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase.

The non-homologous end joining pathway uses pre-existing proteins to repair DNA double-strand breaks induced by ionizing radiation. Here we describe manipulation of this pathway in living cells using a newly developed tool. We generated a single chain antibody variable fragment (scFv) that binds to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in the pathway. In contrast to existing pharmacologic inhibitors, the scFv binds a newly defined regulatory site outside the kinase catalytic domain. Although the scFv inhibits kinase activity only modestly, it completely blocks DNA end joining in a cell-free system. Microinjection of the scFv sensitizes human cells to radiation, as measured by a reduction in efficiency of colony formation and induction of apoptosis at an otherwise sublethal dose of 1.5 Gy. The scFv blocks non-homologous end joining in situ at a step subsequent to histone gamma-H2AX focus formation but preceding gamma-H2AX dephosphorylation. Blockage occurs in cells exposed to as little as 0.1 Gy, indicating that DNA-PKcs is essential for double-strand break repair even at low radiation doses. The ability to modify the radiation response in situ in living cells provides a link between biochemical, genetic and cytologic approaches to the study of double-strand break repair intermediates.

Difference in the induction, but not in the repair, of X-ray- and nitrogen ion-induced DNA single-strand breaks as measured using human cell extracts.

PURPOSE: To compare the repair efficiency of X-ray (low linear energy transfer [LET]) and nitrogen ion (high LET)-induced single-strand breaks (SSB) in a human cell-free end-joining system. MATERIALS AND METHODS: SSB were introduced into a bacterial plasmid, pBR322, by X-rays (4 MeV photons) and nitrogen ions with an LET=125 keV micro m(-1). Repair efficiency was studied under incubation with the protein extracts from human squamous carcinoma cells, UT-SCC-5. RESULTS: A several fold higher dose of nitrogen ion radiation compared with X-ray radiation was needed to induce a similar loss of supercoiled plasmid DNA. There was no difference in the repair efficiency of SSB induced by these two types of radiation. CONCLUSION: The data indicate that X-rays at 25 Gy and nitroging ions at 100 Gy radiation doses, under condition of low scavenging capacity (10 mM Tris), induce SSB of similar complexity or, alternatively, differences in SSB complexity do not alter the repair rate.

DNA damage-dependent inactivation of complementary strand synthesis in Xenopus laevis egg or HeLa cell lysates.

Genotoxic lesions frequently arrest DNA synthesis and, as a consequence, result in the accumulation of incompletely replicated chromosomal segments containing long single-stranded regions of parental DNA. Here, we exploited complementary strand synthesis in Xenopus laevis egg or HeLa cell lysates to test how the eukaryotic replication machinery responds to such damaged single-stranded intermediates. Although both cell lysates promoted efficient conversion of M13 or phi X174 single-stranded templates to double-stranded products, their replication activity was inhibited by DNA damage arising from ultraviolet (UV) radiation or exposure to the alkylating agent N-methyl-N-nitrosourea (MNU). When M13 single-stranded DNA containing UV-or MNU-induced lesions was coincubated with single-stranded substrates containing no lesions, we observed suppression of DNA synthesis on both damaged and undamaged templates. In contrast, complementary strand synthesis remained unaffected in coincubation reactions containing damaged DNA in the double-stranded form. Effective inhibition of complementary strand synthesis on undamaged templates required the presence of at least stoichiometric amounts of UV-or MNU-treated single-stranded DNA, indicating that a DNA polymerase or accessory protein is excluded from DNA synthesis by immobilization at or near the lesion sites. In support of this competitive mode of inhibition, we found that inactivation of DNA synthesis by coincubation with damaged single-stranded DNA was relieved by the addition of an exogenous DNA polymerase that catalyzes processive strand elongation. In summary, this study reveals sequestration of critical components of the eukaryotic replication machinery in a DNA damage-dependent and single-strand-specific manner, thereby providing a potential mechanism to sense arrested replication intermediates during an early recognition step of S phase checkpoint responses.

Comparison of the light-sensitive and cyclic GMP-sensitive conductances of the rod photoreceptor: noise characteristics.

Patch-clamp recordings were made from outer segments of single rod photoreceptors isolated from toad retina. Cyclic GMP (cGMP) and its hydrolysis-resistant analog, 8-bromo-cGMP, increased the conductance of excised membrane patches when applied to the intracellular face of the membrane but not when applied to the extracellular face. The rectification properties of the cGMP-dependent conductance depended on the concentration of divalent cations bathing the intra- and extracellular membrane faces. Current flow was favored from the side of the membrane, either internal or external, that was exposed to bathing fluid free of divalent cations. The power spectral density of cGMP-induced noise was fitted by a sum of 2 Lorentzian components, with corner frequencies differing approximately 15-fold on average. This suggests that channel gating is controlled by 2 kinetic components. To compare the cGMP-sensitive conductance with the light-sensitive conductance of the rod, cell-attached patch-clamp recordings were made from intact, dark-adapted rods, and noise arising from the light-sensitive conductance was analyzed. The power spectrum of the light-sensitive noise also exhibited 2 Lorentzian components similar to those of the cGMP-sensitive conductance in excised patches. These results are consistent with the idea that cGMP opens the light-sensitive channel of the rod outer segment.

Disparity in the production of lymphoblastogenesis inhibition factor by cultured human B and T lymphoid cell lines.

A comparative study of inhibitory effects of cell-free supernatants from cultured human B and T lymphoid cell lines on lymphocyte blastogenesis indicated that the inhibitory effect of supernatant from B lymphoid cells on lymphocyte blastogenesis was significantly higher than that of supernatant from T lymphoid cells or from non-lymphoid neoplastic cells. The inhibitory effect of supernatant was reversible and dose-related. The inhibitory effect gradually diminished with time when the supernatant from B lymphoid cells was added to the culture, 1-3 days after the beginning of cultures. The supernatant of human B lymphoid cells was also found to be highly active in affecting the mouse thymus cell response. The biological nature of this inhibitory factor has not been defined. Both B lymphoid cell lines used in the present study contained Epstein-Barr virus (EBV) genomes while the T-cell line and the non-lymphoid neoplastic cell lines were free of EBV genomes. Sensitivity of the supernatant of B lymphoid cells to u.v. irradiation and heat suggests the possibility that the EBV genomes released into the culture medium may be responsible for inhibition of lymphocyte blastogenesis; resistance of this supernatant to DNase suggests that the EBV genomes may be double-stranded DNA.